USPTO Patents Application 10687850

(19)

J

(12)

Europalsches Patentamt
European Patent Office
Office europeen des brevets EP 0127958 B2

NEW EUROPEAN PATENT SPECIFICATION

(51) Intel 6; G01N 33/48

(45) Date of publication and mention
of the opposition decision:
10.04.1996 Bulletin 1996/15

(45) Mention of the grant of the patent:
11.03.1992 Bulletin 1992/11

(21) Application number: 84303091.7

(22) Date of filing: 08.05.1984

(54) Sensor electrode systems
Sensor-Elektroden
Electrodes pour des detecteurs

(84) Designated Contracting States:
BE CH DE FR GB IT LI NL SE

(30) Priority: 05.05.1983 GB 8312262
05.05.1983 GB 8312261
06.09.1983 GB 8323799

16.12.1983 GB 8333644

11.01.1984 GB8400650
29.02.1984 GB 8405262
29.02.1984 GB 8405263

(43) Date of publication of application:
12.12.1984 Bulletin 1984/50

(60) Divisional application: 89116797.5 89116798.3

(73) Proprietor: MediSense, Inc.

Cambridge Massachusetts 02139 (US)

(72) Inventors:

• Higgins, Irving John
Ravensden-Bedford MK44 2TF (GB)

• McCann, James Michael
London W14 0ES (GB)

• Davis, Graham
Plainsboro-NJ 08536 (US)

• Hill, Hugh Allen Oliver
Oxford OX2 9JH (GB)

• Zwanziger, Ron
Brookline, MA 02146 (US)

• Treidl, Bernhard Ludwig
Boston Mass. (US)

• Birket, Nigel Norman

Sutton Ely Cambridgeshire CB6 2PG (GB)

• Plotkin, Elliot Verne
Bedford, MK43 SG (GB)

(74) Representative:

Dost, Wolfgang, Dr.rer.nat., Dipl.-Chem. et al

Patent- und Rechtsanwalte

Bardehle . Pagenberg . Dost . Altenburg .

Frohwitter . Geissler & Partner

Galileiplatz 1

D-81679 Munchen (DE)

(56) References cited:
EP-A- 0 078 590
DE-A- 3 038 883
US- A- 3 458 421
US-A-4 225 410

DE-A- 2 127 142
US-A- 3 224 436
US-A-4 224 125
US-A- 4 376 689

A.P.F. Turner et al. Biochemical Society
Transactions 11, 445-448 (1983)
H.A.O. Hill et al., Biochemical Society
Transactions 11, 453-455 (1983)
"Medical and Biological Applications of
Electro-chemical Devices" (1980), pp. 6-11
F. Oehme, "Chemische Sensoren" Vieweg
Verlag(1991)

C.C. Liu et al., Diabetes Care 5, 275-277 (1982)
S.J. Updike et al., Nature 214, 986-988 (1967)
"Marine Technology" (1980) 468-472
Review IEE Transactions on Biomedical
Engineering (1978) 479-481

Remarks:

The file contains technical information submitted
after the application was filed and not included in
this specification

Primed by Jouve. 75001 PARIS (FR)

EP0127 958 B2

Description

THIS INVENTION relates to sensor electrode sys-
tems.

Sensor electrode systems are known, for example,
from DE-2127142, EP 78590 and US 4224125. The
present invention is concerned with sensor electrode
systems of the kind generally known from US 4224125.
Our European Patent Application 82305597 (EP-A
78636) describes the construction of sensors comprising
a conductive electrode coated with a mixture, or layers,
of a catalytically active enzyme and a mediator com-
pound and usually further coated with a retaining perme-
able membrane. When such a coated electrode is con-
tacted with a substrate containing a species for which
the enzyme exerts a catalytic effect, the mediator com-
pound transfers charge to the electrode and this can be
used to give a readout signal, against a standard elec-
trode, correlated with the concentration of the said spe-
cies, even in the presence of other species since en-
zymes are typically highly selective in their catalytic ac-
tion.

Thus, numerous types of enzyme-coated electrodes
have been utilised each specific to the presence of a
physiological or other substrate for which the particular
enzyme acts as a catalyst and each therefore potentially
capable of acting to detect, measure or monitor the level
of the substrate in vivo or in vitro and give a readout
correlated for instance with an underlying physiological
condition controlling or affecting the substrate level. In
particular, use of glucose oxidase or bacterial glucose
dehydrogenase as the enzyme, associated with suitable
electron-transferring mediator compounds, has been
shown to give readout signals correlating linearly with jn
vitro blood glucose levels over a wide range thus giving
a diagnostic or measuring tool for diabetic conditions. Al-
so, such electrodes can measure glucose levels in plas-
ma, serum, interstital fluid, saliva or urine.

The mediator compounds described in EP-A 78636
include polyviologens, fluoranil and chloranil. However,
the preferred mediator compounds are metallocene
compounds, and in particular the ferrocenes (biscy-
clopentadieny! iron and its derivatives).

The particular advantages of ferrocenes are as fol-
lows: -(a) a wide range of redox potentials accessible
through substitution of the cylopentadienyl rings (b) func-
tionalisationof the rings, e.g. to confer solubility or chem-
ical linkability to other such rings or other system com-
ponents (c) electrochemically reversible one-electron re-
dox properties (d) pH -independent redox potential and
(e) slow autooxidation of the reduced form.

The ferrocene structure may be modified by substi-
tution on the rings, and/or by association or polymerisa-
tion, which modifications affect the physical, chemical
and electrical behaviour so that optimisation of a partic-
ularsensor electrode material is possible. In general use,
the compound 1,1'dimethylferrocene is a valuable medi-
ator.

Copending Application EP 84303090. 9 of even date
herewith entitled "Assay techniques utilising specific
binding agents" is concerned with the effect on the en-
zyme and/or mediator electro chemical availability of

5 specific binding agents e.g. antigens/antibodies and oth-
ers. It can be embodied by specialised electrodes. Such
electrodes fall within the scope of the present invention
and are discussed below. The interested reader is re-
ferred to the published specification, EP-A 125139.

10 The prior art Application EP-A 78636 referred to
above discloses equipment utilising such sensor elec-
trodes. In general it is suitable for research or institutional
(e.g. hospital) use. The present invention is concerned
with providing sensor electrodes and equipment for use

*5 by lab persons or without extensive technical back-up
services.

US-A-4 225 410 describes a sensor electrode sys-
tem for measuring or monitoring glucose in a liquid mix-
ture by monitoring the anodic current resulting from the
20 electro-oxidation of hydrogen peroxide produced when
a glucose oxidoreductase enzyme catalyses a redox re-
action of glucose. Alternatively, anodic charge is meas-
ured. The sensor electrode system comprises a throw
away test electrode carrier carrying an area of working
& electrode material comprising the glucose enzyme, the
area being adjacent to but non -contiguous with an area
of reference electrode material, both electrode areas be-
ing of small dimension and extending as or supported on
the electrode carrier to facilitate manipulation before or
30 during contact with a small withdrawn sample of blood.
US-A-4 376 689 describes enzyme electrodes
which contain an oxidore-ductase enzyme and a corre-
sponding coenzyme, such as NAD + or NADP+, in addi-
tion to an electron-conducting material, such as graphite.
35 The coenzyme serves as a transferor of electrons be-
tween enzyme and electrodes and vice versa.

"Medical and Biological Applications of Elec-
tro-chemical Devices" (1980), pages 7-11, contains
general comments concerning the application of meth-
40 ods termed amperometry, voltammetry and polarogra-
phy in medicine and biology.

F. Oehme, "Chemische Sensoren", Vieweg Verlag
(1991), pages 76/77, contains a statement that amper-
ometry is a method belonging to voltammetry.
45 C.C. Liu et aL, Diabetes Care 5, 275 - 277 (1982)
describes a miniaturized sensor for determining oxygen
wherein a reference electrode of silver/silver chloride is
located adjacent to a working electrode made from gold
on a flat elongate carrier material made from aluminum.
50 The possibility of using such oxygen sensors as throw
away sensors is addressed.

S.J. Updike et al., Nature 214, 986 - 988 (1967) is
cited as a reference in C.C. Liu et al. for the proposition
that measurements of glucose in blood or extracellular
55 fluid can be accomplished by measuring the oxygen lev-
els in the presence of an enzymatic catalyst, glucose ox-
idase, by using two oxygen sensors.

"Marine Technology", (1980), pages 468 - 472 de-

2

3

scribes a dissolved oxygen sensor comprising an anode
of the silver/silver chloride type and a cathode of the gold
type used for obtaining data in numerous potential bio-
logical applications and particularly in oceanographic ap-
plications. 5

Use may be made of such electrodes in chemical
industry especially where complex mixtures are encoun-
tered, e.g. in food chemistry or biochemical engineering.
They are however of particular value in biological inves-
tigation or control techniques, in human or animal med- 10
icine.

We have now established certain design criteria in
the production of such electrodes for lay, or clinic, use.

The electrodes can be used in an invasive probe
( i.e. one which enters body tissue to contact a body fluid '5
such as whole blood or subcutaneous tissue fluid) or as
part of an external test upon a withdrawn sample (using
a syringe) or upon an expressed sample (e.g. using a
needle-prick device). In each instance the electrode
must be as small as practical to avoid trauma either on 20
invasion of the tissue or withdrawing of the sample.

It must be elongate, either to fit within a pointed nee-
dle, or for ready handling as an electrode for ready as-
sembly to equipment on the one hand and contact with
the sample on the other. It must be sensitively manipu- 25
lable. It must carry, prior to assembly or in the assembly
structure, the reference electrode as well as the 'sensi-
tive' electrode, in spaced non-contiguous relationship.

The present invention provides a sensor electrode
system of the kind for use in measuring or monitoring 30
glucose in a liquid mixture, by monitoring the flow of cur-
rent when a glucose oxidoreductase enzyme catalyses
a redox reaction of glucose, and a mediator compound
transfers electrons between an electrode and the cata-
lytically active oxidoreductase enzyme; characterised in 35
that the sensor electrode system comprises a throw a
way strip test electrode carrier earring an area of working
electrode material comprising the glucose enzyme and
ferrocene or a ferrocene derivative at the mediator com-
pound, the area being adjacent to but non-contiguous *o
with an area of reference electrode material, both elec-
trode areas being of small dimension, and extending as
or supported on the electrode carrier formed as an elon-
gate member to facilitate manipulation before or during
contact with live tissue or with a small withdrawn sample 45
or body fluid, and wherein said working electrode mate-
rial is a single layer admixture of said glucose enzyme,
said ferrocene mediator and conductive carbon.

Sensor electrode systems of this invention can be
dipped into or similarly contacted with a liquid substrate so
e.g. a glucose-containing small blood sample or drop of
blood.

In one aspect of the invention said sensor electrode
system comprises separate electrical connection to each
electrode for attachment to a read-out means denoting 55
amount, or monitored level of glucose in a liquid medium
with which the support member is contacted to contact
both electrodes.

4

The elongate carrier conveniently comprises a flat
strip.

The first electrode is preferably formed of carbon
e.g. a filter paper containing carbon. We have also found
that carbon foil e.g. as available under the Trade Marks
"GRAPHOIL" or "PAPYEX" is a valuable electrode ma-
terial. As to the glucose oxidoreductase enzyme, the use
of glucose oxidase or dehydrogenase, e.g. the bacterial
glucose dehydrogenase from Acinetobacter calcoace-
ticus is particularly valuable. The mediator compound is
ferrocene or a ferrocene derivative (especially
1 , 1 'dimethylferrocene).

By way of example only, carbon foil can be glued to
the strip; 1,1 '-dimethylferrocene mediator can be depos-
ited on the surface of the foil by evaporation of a toluene
solution; and enzyme can be bonded to the surface by
the use of 1-cyclohexyl-3-(2-morpholinoethyl) carbodi-
imide metho-p-toluene sulphonate (referred to below as
"carbodiimide w ).

The second electrode can be any convenient refer-
ence electrode. We have found it useful to provide adja-
cent but not contiguous to the first electrode, a flat layer
of silver and to convert the surface thereof to silver chlo-
ride so as to give an Ag/AgCI reference electrode.

Typically, the electrical connections can be wires
which extend down, and are preferably adhered to, the
strip, and make electrical contact each with its respective
electrode.

The readout means is preferably a digital indicator
suitably connected to a dedicated potentiostat which
poises the carbon electrode potential at e.g. +150mV
Ag/AgCI for a glucose system. The current flowing is then
proportional to glucose concentration.

In a particularly valuable version of this type of sen-
sor, it comprises (a) a flat first electrode area of known
area small enough to be completely coverable by the
smear of blood produced from a non-expressed drop of
blood generated from a needle-prick at a bodily extrem-
ity, (b) a reference electrode area on the same surface
separate from but sufficiently close to the sensitive elec-
trode area that the said blood smear also reaches the
reference electrode to establish electrical communica-
tion and (c) conductive elements extending separately
along the same surface of, and thus insulated from the
elongate support member, communicating one with each
electrode for connection to signal read-out means at-
tachable to one end of the member.

The area of the first (i.e. sensitive) electrode is gen-
erally substantially square; it may be rectangular or oth-
erwise shaped, but in any case usually will correspond
in area to a square of 5 mm edge length, or below e.g.
from 2 to 4 mm. Equipment utilising the sensors of this
invention can be portable or "desktop".

In one form, equipment utilises such electrodes to
give visible readout correlated with a selected physiolog-
ical parameter thus being capable of use in human or
veterinary medicine by medical or nursing personnel, or
by experienced lay subjects on a self-measurement ba-

EP0127 958 B2

3

EP 0 127 958 B2

6

For convenience, this document will refer hereinaf-
ter to blood-glucose-measuring equipment as being typ-
ical but not limitative of equipment with which the sensor
electrode systems of the present invention can be used.

Diabetic subjects need to measure their glucose lev-
els frequently. Hitherto, a common method carried out
by the subject personally is a colorimetric test using a
blood or urine sample which is applied over a surface
area containing a colour-reactive detector chemical, ad-
jacent to a comparison area, to give a colour change
which is compared with a chart of colour values as an
approximate measure of glucose level.

There are however defects in this method. Firstly,
colorimetric changes are quantitatively difficult to as-
sess, especially if the patient has impaired vision as a
result of the diabetic condition. Indeed, because of this
problem expensive automatic colour comparison equip-
ment may need to be purchased by some subjects for
interpreting the test results. Secondly, the blood test,
while inherently more accurate than a urine test, needs
a large enough sample to cover the test surface. Thirdly,
it requires the patient to time the colour development ac-
curately. Since blood samples, on a self -treatment basis
are taken from body extremities (fingers, toes, earlobes)
they are normally not large enough when obtained by a
simple needle-prick, and must in fact be expressed i.e.
squeezed or massaged out to form a larger drop. Pro-
gressively, the tissue of the extremities becomes scarred
and coarsened by such treatment to an extent whereby
finding fresh testing sites presents a problem.

In order to embody the invention on a home-diag-
nostic basis a main object of the present invention in one
aspect is as described above the provision of small scale
non-traumatic test pieces, as an external test electrode
strip capable of using the naturally-arising small blood
droplet from a needle-prick tester, without tissue mas-
sage. Examples are described in more detail below.

These small-scale electrodes are intended as sin-
gle-use throwaway articles and are utilised in conjunc-
tion with electrical circuitry and a readout means, to
which they must be easily attachable and detachable.
Such circuitry and readout means is itself preferably em-
bodied on a very small scale.

We have accordingly found that the totality of the
equipment is subject to certain design constraints.

Thus it is a further object of the invention in this form
that the device should be non-traumatic to the user either
physically e.g. if used with its own invasive probe or psy-
chologically by virtue of its appearance.

It is a further object of the invention in this form that
the device should be capable, despite the small size of
the throwaway electrode and of the permanent circuit-
ry/readout components, of easy assembly and disas-
sembly even by juvenile or elderly lay users.

It is still a further object of the invention in this form
to ensure that the relatively expensive permanent circuit-
ry/readout components should, despite their small size,

be of a form which minimizes loss or damage.

It is still a further object of the invention in this form
to provide a device the display readings of which are vis-
ible and understandable to a non-expert user.
5 We have now found that these and other objects of
the invention can be met by assembly of the circuitry/re-
adout components into a housing resembling a pen or
digital-watch.

Thus, for use with sensor electrode systems of the

10 present invention, there is provided an assembly of cir-
cuitry and display means for use in producing a readout
value as a diagnostic aid in human or veterinary medi-
cine, housed in a pen- like hollow elongate housing hav-
ing (a) at one end an electrically conductive socket suit-

15 able to receive the outer end of the sensor electrode sys-
tem capable of producing an electrical signal correlating
with a physiological parameter to which the sensor elec-
trode system is selectively sensitive and (b) towards the
other end a digital read-out window for exhibiting a nu-

20 merical value corresponding tothe parameter. A thermis-
tor may also be used for temperature compensation.

The person skilled in the art of designing medical
equipment will appreciate that use of the invention ex-
tends to the pen-like assembly in combination with an

25 attached sensor electrode system as test member, and
to the combination as a kit of interrelated parts of such
an assembly with a plurality of test members suitable for
one-off use.

The term "pen -like" is a general limitation on size

30 and shape. In functional terms, its characteristics are
such that it can be held near the socket between the
thumb and the nearer one or two opposed fingers, with
the elongate body resting on and extending beyond the
forefinger, but not to an extent that prejudices fine control

35 of the socket end by the thumb and fingers. In numerical
terms it can be from 1 0 to 30 cm. long and from 0.5 to 3
cms across its maximum transverse dimension; more
usually it will be from 12 to 20 cms. long and from 0.8 to
1.5 cms. across. It can be generally circular, or polygo-

40 nal, in cross-section. Each detachable test member is
usually a small-scale enzyme-coated sensor electrode,
of the type discussed in the earlier Patent Applications
listed above, where the enzyme is specifically glu-
cose-catalyzing whereby diabetic conditions can be

45 measured. It may be a flat external strip electrode dimen-
sioned to operate on a small, non-expressed, blood
droplet. The socket arrangement will vary accordingly.

In one embodiment of the present invention, two or
more sensor electrodes may be incorporated into a sin-

so g|e test member. Again, the socket arrangement will vary
accordingly.

The readout means will typically be a conventional
seven-segment display window towards the rearward
end of the "pen" as in conventional pen/watches. In the
55 case of the multiple sensor embodiment described in the
preceding paragraph the display may be switchable be-
tween each sensor's discrete monitoring circuit, both the
display and a single monitoring circuit may be switchable

4

7

EP0 127 958 B2

8

between sensors, or, a specific display may be provided
for each of the sensors present.

Such equipment is of course particularly adapted for
use with the non-invasive strip sensor defined above.

Another form of equipment for use with the sensor
electrode systems of the present invention is so-called
"desk-top" equipment, i.e. for general but skilled use in
a general clinic.

In this aspect, the aim is to provide suitable equip-
ment for a practitioner of "desk-top* scale and complex-
ity.

In the operation of a glucose sensor a number of rel-
evant technical points and advantages should be con-
sidered. These features are:-

I Constructional Features

fa) Membrane cover for electrode

Although the enzyme electrode should be in electri-
cal contact with the liquid, it may be found valuable to
exclude the sensor from interfering contact with larger
molecules or tissue fluid components. This can be done
by a covering or surrounding membrane, depending on
electrode geometry. Heat-shrinkable thin polymer tubing
can be used as, or in connection with, such membranes.

The membranes can be polymerised in situ (e.g. cel-
lulose acetate). A particular valuable membrane is
formed by polycarbonate, especially those polycar-
bonates sold under the Trade Marks "NUCLEOPORE"
or "STERILIN". When tissue fluids are examined they
may contain ascorbate; polycarbonate membranes do
not permit the passage of ascorbate and thus virtually
eliminate interference from that substance. Alternatively
a polyurethane membrane may be employed.

(b) Type of carbon

Carbon foil, as strips, or carbon attached to metal
meshes, of pyrolytic grade and known by the Trade
Marks "GRAPHOIL" and "PAPYEX" are much preferred
for carbon-ferrocene electrodes for use with glucose ox-
idase. Oxygen interference is minimal, there being less
than 4% change in signal between anaerobic and fully
aerobic samples. Their physical nature is also very con-
venient for fabrication, especially of small-scale devices.

II Operational features

fa) Operational potential

Preferably operation should take place at a potential
equivalent to +50 to +200 mV vs. SCE since interference
caused by oxidation of other chemical species present
is thereby reduced.

fb) Concentration range

Glucose oxidase can be used to monitor glucose
concentrations of 0 to 40 mM, and glucose dehydroge-
s nase at 0 to 20 mM when immobilised on a carbon-fer-
rocene electrode. The sensor response is linear up to
about 40 mM.

fc) Response times

w

The glucose oxidase sensor without membrane is
kinetically limited giving rapid response times i.e. about
20 seconds to 95% of the steady-state current response.

'5 (d) Oxygen-sensitivity

Glucose dehydrogenase/ferrocene electrodes are
totally oxygen -insensitive.

In practice, a realistic device can achieve good per-
formance without a third electrode, using Ag,AgCl as a
reference counter-electrode, as described more fully be-
25 low.

ff) pH and temperature

Glucose oxidase electrodes show no change in cur-
30 rent output between pH6 and pH9, and are thus relatively
pH-insensitive. They are temperature-stable up to 40°C.
If necessary temperature compensation can be effected
using a thermistor, or a constant temperature jacket may
be used. Also, operating with the electrodes diffu-
35 sion-limited minimises temperature effects.

Electrodes may be stored moist. Extended storage,
40 over months or years, may be achieved by f reezedrying
or air-drying.

Finally, some aspects can be considered of the elec-
trical circuitry for operating the equipment as described.
The sensor electrode systems of this invention can
45 be employed with a measuring device comprising means
for comparing an electrical output of the electrode sys-
tem with an electronic reference and means for providing
a signal related to the electrical output of the electrode.
By employing an electronic reference rather than a
50 cell or reference electrode a measurement using a sen-
sor including an electron-transfer electrode may be
made without the use of a separate electrode as a refer-
ence.

In a preferred embodiment of the invention, the elec-
55 tron-transfer electrode is poised at a fixed potential
against a reference electrode, and the current flowing in
the electron-transfer electrode is measured.

The invention will be further described with refer- .

20 fe) Use of third electrode

30
35

fg) Storage of Electrodes

9

EP0127 958 B2

10

ence to the accompanying drawings. In these drawings
the actual description of Figures 1/2 and of Figure 6 is
not an embodiment in accordance with the invention for
the reason that the working electrode material (of elec-
trode 4 in Fig. 1/2 and of electrode 31 in Fig. 6) is not a
single layer admixture of glucose enzyme, ferrocene me-
diator and conductive carbon. However, on the one hand
these Figures are helpful in understanding the invention
and, on the other hand, the embodiments of Figures 1/2
and 6 represent in fact embodiments of the invention if
the electrode construction is carried out as described at
the very end of the description under the headline 7n-
ventive embodiment for the electrode manufacture".
In the following drawings

Figure 1 is a front view of a strip-supported electrode
configuration;

Figure 2 is a back view of the combination shown in
Figure 1 ;

Figure 3 shows an alternative strip-supported elec-
trode;

Figure 4 shows a strip-supported electrode which is
a variant of Figure 3;

Figure 5 shows a modified connection of the strip
electrode of Figures 3 and 4;
Figure 6 shows a further alternative supported elec-
trode;

Figure 7 shows a combination of two electrode sup-
ports;

Figures 8a and 8b are general diagrammatic side
views of a pen-like portable holder, of particular util-
ity for the electrodes shown in Figures 3, 4 and 5,
having an assembly of circuitry and having a
read-out window;

Figure 9 shows a schematic diagram of one form of
electrical circuitry for use with the electrodes and
equipment of the present invention;
Figure 10 shows a more elaborated circuit diagram
for use in the embodiment of Figure 9;
Figure 1 1 shows a schematic diagram of an alterna-
tive embodiment of electrical circuitry; and
Figure 12 shows the more elaborated circuit dia-
gram of a yet further embodiment of circuitry.

In the following description of Figures 1 and 2 dimen-
sions, materials amount and proportions are given by
way of example only.

A strip of epoxy glass 1 , 9.5 x 40 x 1 .6 mm, has two
1 mm diameter holes 2 and 3 therein. A 9 x 9 mm piece
of graphite tape or foil 4 is glued on one face, near the
end to cover hole 2 and a 4 x 9 mm strip of silver foil 5
is glued adjacent thereto over hole 3. Wires 6 and 7 (Fig.
2) on the back enter holes 2 and 3 respectively for elec-
trical connection with the respective electrode material
4 and 5, being glued in the holes by conductive epoxy
resin 8. A stabilising layer of epoxy resin is present over
at least part of the back e.g. at 9 to keep the wires in
place. Carbon electrode 4 is covered with 1, V-dimeth-

w

15

ylferrocene and glucose oxidase. Silver electrode 5 is
covered with silver chloride.

The strip is made up in the following sequence:-

(a) drill holes 2 and 3,

(b) glue on electrodes 4 and 5; "ARALDITE" epoxy
resin is suitable but should not enter holes 2 and 3,

(c) attached wires 6 and 7, using conductive epoxy
8, and apply "ARALDITE" resin at 9 the fix the wires
in place,

(d) hold the silver electrode at +400 mV vs. SCE in
5M chloride for 10-15 seconds to deposit a thin AgCI
layer,

(e) apply a solution of 1,r-dimethylferrocene (4 |il)
in toluene (20 mg/ml) to the graphite tape 4 and allow
to evaporate,

(f ) cover the ferrocene-coated tape with 50 u.l of car-
bodiimide (25 mg/ml) in pH 4.5 acetate buffer for 1
1/2 hours and

20 (g) rinse and cover with glucose oxidase (12.5
mg/ml) in pH 5. 5 acetate buffer for 2 hours.

The strip can be used by attaching wires 6 and 7 to
a potentiostat poising the potential at electrode 4 at +1 50
25 mV. vs. Ag/AgCI, and dipping the strip into a glu-
cose-containing solution so that both electrodes 4 and 5
are covered. The shape of the strip facilitates such han-
dling. The current flowing is proportional to glucose con-
centration.

30 Figure 3 shows a strip electrode 1 7 made of, for ex-
ample, a ceramic material or printed-circu it-board lami-
nate. It includes a square area 18 with connector lead
1 9, the square being covered with the enzyme-contain-
ing layers as described above. It further includes a small
35 reference electrode area 20 and separate connector
lead 21. The rearward end 22 of the electrode 17 fits into
a socket as shown in Figures 8a and 8b and described
below. It is to be noted that, as with the needle 10, the
electrode strip 17 is a small-scale device. Thus square
40 area 18 is of a side length only about half that of each of
two square colorimetric test areas of conventional diag-
nostic tests and can be used with the original non-ex-
pressed bead of blood from a needle-prick device, which
is adequate to cover the whole of the square area and
45 communicates electrically with reference electrode area
20,

Modifications may be made to the embodiments
shown in Figure 3. For example the strip electrode as
shown in Figure 3 can be longer, whereby electrodes 18
50 and 20 are located only partway along the strip, leaving
a free end 22a to facilitate ease of handling by the patient
without damaging or touching the electrodes. Also, the
electrode strip can clip within two opposed contacts or
resilient mounting 31 , the routing of one or other of con-
55 ductive lines 19 and 21 being modified accordingly.

Figure 4 shows a longer strip, and Figure 5 shows
the inner end of a strip held between two resilient metal
contact strips.

6

11

EP0 127 958 B2

12

In Figure 6 a 2 cm length of eleclrically insulating
polymer for example MYLAR or TEFLON (a polyfluoro-
carbon) 0.3 mm square in transverse cross-section is
provided with a palladium-silver conductive electrode 31 ,
on the front surface as shown, and a second, smaller
electrode 32, on the back as shown in dotted lines. In
each case conductive lines 33 and 34 respectively, were
formed simultaneously with the electrodes.

On the front electrode 31 is painted a mixture of tol-
uene, 1,1'-dimethyl ferrocene and graphite, formed by
mixing a solution of the toluene and 1 r 1'-dimethyffer-
rocene and a slurry of toluene and graphite. It is believed
that the ferrocene is adsorbed on to graphite particles.
After drying the mixture forms a layer 35. A layer 36 of
glucose oxidase is then immobilised on the graphite sur-
face by carbodiimide immobilisation, known per se (en-
zyme adsorption can also be used). The electrode may
then be covered, on both sides, with a semipermeable
membrane of cellulose acetate (or polyurethane), not
shown, to block large interfering species from contact
with the electrode.

The square section of the support helps in the paint-
ing of slurry, or the enzyme-attachment stages, in keep-
ing the electrodes 31 and 32 distinct.

The small scale electrode so produced could be
used per se but is especially valuable for incorporation
into a standard gauge needle, giving a blood-glucose
reading using the same invasive member as the eventual
injection.

When producing such small scale needles, the exact
sequence of steps can vary. For example, graphite could
first be painted on, and a solution of the mediator (fer-
rocene, etc) in toluene then be applied by dipping into
the graphite. Likewise, the enzyme can be applied in so-
lution for adsorption. There is therefore a danger that the
reference electrode e.g. silver/palladium could be ad-
versely affected by the solvents or solutes used.

Figure 7 shows the key steps of a procedure which
can be used to advantage in the fabrication of these mi-
croelectrodes.

The reference electrode 37 and its conductive
lead-out strips 38 are formed in silver/palladium on TE-
FLON base 39. Similarly, an electrode support 40 and
lead-out strip 41 are formed in silver/palladium on TE-
FLON base 42. Only this base 42 and its electrode sup-
port are then subjected to (a) painting on a graphite slurry
in toluene (b) dipping in 1',1'dimethylferrocene solution
in toluene and (c) contacting with the enzyme to absorb
e.g. glucose oxidase into the active layer 43. Thereafter
the bases 39 and 42 are glued or held, side-by-side, their
general rectangular cross-section facilitating such posi-
tive location. Back-to back location is also possible.

As before, the finished assembly may be located in-
side a needle bore, e.g. with extra access portions near
the electrode surfaces.

Incorporation of 1M'-dimethvlferrocene into the
electrode

A solution of 1',1'dimethylferrocene in toluene was

5 mixed into a toluene-based slurry of the graphite powder.
The mixture was then painted on to the base conductor
41 and allowed to dry at 43. This provided an electrode
surface that was electroactive towards glucose oxidase.
These experiments showed that "thick layer'or

10 screen -printing technology could provide a usable base
strip which could easily be coated with a stable graphite
surface and that moreover the electrode surface could
be made electroactive towards glucose by adsorption of
a ferrocene directly into the coating mixture. In addition,

15 the reference electrode operated satisfactorily in buff-
ered solutions.

Figures 8a and 8b show a holder which is particularly
adapted to utilise electrodes as shown in Figures 3, 4, 5
but which could if necessary utilise electrodes as shown

20 h Figures 1 and 2, and 6 and 7 at least of the various
embodiments shown.

From above the holder 81 intentionally resembles a
conventional pen/watch as much as possible. It has a
forward end 82, possibly rotary to tighten the walls of a

25 flattened socket cavity 83 formed within it. A central join,
a clip 84 and a press-button 85 all resemble those of a
conventional pen, and digital readout-window 86 is also
of a type known in pen/watches.

Inside the holder as shown by dotted lines is con-

30 nection circuitry 87, possibly printed in situ, battery 88
and operating circuitry 89 behind and manufactured as
a unit with the display window 86. The display can be
capable of operation only when button 85 is pressed so
that extra illumination can be provided if necessary,

35 The embodiments shown in Figures 8a and 8b es-
pecially when used in conjunction with the electrodes of
Figures 3 - 5 fulfill the design criteria discussed above
for such portable equipment.

The delicate manipulation facilitated by the pengrip

40 (e.g. by thumb and finger) means that the small elec-
trodes e.g. of Figures 3, 4 or 5 can be easily assembled
into, or detached from, the socket. A user will always ori-
ent the holder with the window 86 visible thus always giv-
ing a uniform relative orientation to the socket 83 where-
as by the rearward ends of the fragile electrodes can be fit-
ted without experiment and damage.

The "pen" format is instinctively picked up after use
and safely carried in a pocket, more so than for any other
small device. Thus the expensive part of the equipment

50 is safeguarded. Furthermore, it is possible to incorporate
a conventional timer circuit into the device thereby fulfill-
ing the actual function of a pen-type watch and providing
an audible or visible signal which marks the point in time
at which a reading should be taken.

55 Finally, the display is numerical, clearly visible and
if necessary can be supplemented by an illuminating light
source.

In Figure 9, a sensor 101 is connected between a

7

13

EP0127 958 B2

14

voltage buffer 1 02, and the inverting input 1 03 of the op-
erational amplifier 104 which is configured as a current
amplifer. An electronic reference 105 connected to the
non-inverting input 106 of the operational amplifier is fed
into a low-pass filter 1 07 which removes rapid signal fluc-
tuations (which may be due to noise, earth hum or other
sources of interference) while allowing the filtered output
of the operational amplifier 104 to be fed into the digital
volt meter (D.V.M.) 108.

The digital volt meter is supplied with clock pulses
via the divider 1 09, from the timer 111. The D.V.M drives
a liquid crystal display 1 1 2. The electronic reference 1 05,
is either of a pre-selected value or capable of being se-
lected for a particular electron transfer electrode.

In each of the embodiments of electrode discussed
above the sensor comprises a mediator-carrying surface
which has one or more enzymes immobilized thereupon.
The sensor further includes a silver/silver chloride
(Ag/AgCl) internal reference electrode. If, for example, a
voltage of + 200 mV volts is preferentially dropped
across the electrode as is the case with a glucose -oxi-
dase-containing electrode, then the reference voltage
1 05 is selected accordingly.

In Figure 10. the electronic reference comprises re-
sistors 152, 153 and 154 together with diodes 151. Avoft-
age is selected at 0.3V by a suitable choice of resistor
values at 152, 153 and 154. The voltage across the re-
sistor 153 is 1V, which ensures that 100 u.A of electrode
current will give a full scale reading of 999 on the liquid
crystal display 109.

Non-inverting buffer 102 ensures that the voltage on
the terminal 101 A remains substantially constant The
sensor 1 (Figure 1) is connected across terminals 101 A
and 101B.

The operational amplifier 104 is connected via its in-
verting input 103 to the terminal 101 Band the feedback
resistor 141 . The non-inverting input 106 is connected to
the electronic reference.

A first-order low-pass filter 171, 172 is connected
across the feed-back resistor 141, and supplies an ana-
log signal to the D.V.M. 8. Pin values are given for a
7116CPL chip (manufactured by Motorola). The D.V.M.
drives a liquid crystal display 112.

Clock pulses for the D.V.M. are supplied from the
timer 111, (pin values are given for a 555 chip) via the
dividers 1 91 (pin values for an MC 1 4020B) and 1 92 (pin
values for an MC14016B chip). The connection 193 to
pin 11 of the divider 191 enables a power-up reset

In Figure 11 an alternative circuit is shown which
does not make use of the non-inverting voltage buffer
102, but has the sensor 101 connected between the op-
erational amplifier 104, and ground. The embodiment
shown in Figure 11 employs a fixed reference voltage
which is provided by a circuit differing from that of Figure
10.

In this embodiment, the diode 155 functions as a
voltage-reference diode and provides a reference volt-
age drop across its ends equal to the diode forward volt-

age.

By a suitable choice of the values of the resistors
156, 157 and 158 the correct voltage may be applied
across the electrode. In this embodiment the sensor
5 again employs as a reference an Ag/AgCl couple and
immobilized glucose oxidase in the presence of a medi-
ator compound as the electron-transfer electrode.

Figure 12 shows a third embodiment of the present
invention, which provides a continuously variable refer-
70 ence voltage which may be selected to accommodate
any type of electron-transfer electrode, that is, one,
which for example, employs any of the enzymes listed
herein or any combination of these enzymes. The LED
display is not shown.
'5 in Figure 12 the feedback resistor 141 in circuit at
any given time may be selected from resistors 141a,
141b, and 141c by means of switch SW2a which is
ganged with switch SW2b. This allows the current output
of the sensor 101 to be displayed in three ranges, for
20 example, 1 u,A 10jiAor 100 \iA full scale. Furthermore,
the range may be trimmed by using the variable resistors
142a and 142b.

The embodiment shown in Figure 12 has the non-in-
verting voltage buffer 102 of the embodiment shown in
25 Figure 10.

The reference voltage for the embodiment shown in
Figure 12, is derived from the circuit elements 501-505
which include the potentiometers 503 and 502 providing
a variable voltage across the sensor 101, thereby ac-
30 commodating any type of electron-transfer electrode. It
is envisaged that the continuously variable resistors
142a and 1 42b could be replaced in certain applications
by stepwise resistance switching means with each posi-
tion or setting being dedicated to a particular type of elec-
ts trode.

Various modifications may be made in the circuitry.
For example the liquid crystal display may be replaced
by a plotter or a dosage control device, or a temperature
stability circuit may be incorporated.
40 Inventive embodiment for the electrode manufac-
ture.

The electrode is manufactured by screen printing
techniques e.g. in a multi-stepped procedure compris-
ing:-

45

I - screen printing of Ag/AgCi reference electrode
and metal tracing.

II - screen printing of the active electrode with a print-
ing ink comprising a colloidal carbon, glucose oxi-

50 dase in buffer, and an organic polymer.

ill - screen printing, spraying or dip coating to pro-
vide a membrane over the assembly.

Advantages of this method are that it is amenable to
55 high volume automation, and is of high reproducibility

To this end, there can be used a suspension in a
liquid medium of carbon together with (a) glucose oxi-
dase as enzyme and (b) ferrocene or a ferrocene deriv-

8

15

EP0 127 958 B2

16

ath/e as mediator compound capable of transferring
charge to the said carbon from the enzyme when the en-
zyme is catalytically active, the said suspension being
formed as a printable and conductive ink for use in the
fabrication of electrodes as described above. 5

Claims

1. A sensor electrode system of the kind for use in 10
measuring or monitoring glucose in a liquid mixture

by monitoring the flow of current when a glucose oxi-
doreductase enzyme catalyses a redox reaction of
glucose, and a mediator compound transfers elec-
trons between an electrode and the catalytically is
active oxidoreductase enzyme; characterised in that
the sensor electrode system comprises a throw
away strip test electrode carrier earring an area of
working electrode material comprising the glucose
enzyme and ferrocene or a ferrocene derivative as 20
the mediator compound, the area being adjacent to
but non -contiguous with an area of reference elec-
trode material, both electrode areas being of small
dimension, and extending as or supported on the
electrode carrier formed as an elongate member to 25
facilitate manipulation before or during contact with
live tissue or with a small withdrawn sample of body
fluid, and wherein said working electrode material is
a single layer admixture of said glucose enzyme,
said ferrocene mediator and conductive carbon 30

2. A sensor electrode system according to claim 1 .
comprising separate electrical connection to each
electrode for attachment to a read-out means denot-
ing amount, or monitored level of glucose in a liquid 35
medium with which the support member is contacted

to contact both electrodes.

3. A sensor electrode system according to claim 2,
which comprises (a) a flat first electrode area of 40
known area small enough to be completely covera-

ble by the smear of blood produced from a
non-expressed drop of blood generated from a nee-
dle-prick at a bodily extremity, and (b) a reference
electrode area on the same surface separate from 45
but sufficiently close to the sensitive electrode area
that the said blood smear also reaches the reference
electrode to establish electrical communication and
(c) separate conductive elements extending along
the same surface of the elongate support member, so
communicating one with each electrode for connec-
tion to signal read-out means attachable to one end
of the member.

4. A sensor electrode system according to claim 1,2 ss
or 3 in which the area of working electrode material

is square.

5. A sensor electrode system according to any of
claims 1 to 4, in which the area of working electrode
material is less than 25 mm 2 .

6. A sensor electrode system according to any preced-
ing claim, in which the reference electrode is sil-
ver/silver choride.

7. A sensor electrode system according to claim 1,
wherein the working electrode and the reference
electrode are carried on the same face of an elon-
gate non-conductive carrier of rectangular
cross-section.

8. A sensor electrode system according to claim 7, in
which the area of the working electrode material is
towards one end of the surface of the elongate mem-
ber, and the area of the reference electrode is also
towards the said end.

Patentanspruche

1. Sensor-Elektrodensystem zur Verwendung beim
Messen oder Uberwachen von Glukose in einerflus-
sigen Mischung, durch Uberwachen des Stomflus-
ses bei einer Redoxreaktion von Glukose, die durch
ein Glukose-Oxidoreduktase-Enzym katalysiert
wird, wobei eine Vermittterverbindung Elektronen
zwischen einer Elektrode und dem katalytisch akti-
ven Oxidoreduktase-Enzym Obertragt, dadurch
gekennzeichnet, daft das Sensor-Elektrodensy-
stem einen wegwerfbaren Streifentest-Elektroden-
trager aufweist, der eine Flache aus Arbeits-Elektro-
denmaterial tragt, welche das Glukoseenzym und
Ferrocen oder ein Ferrocender'rvat als Vermittlerver-
bindung umfaBt, wobei diese Flache benachbart zu
einer Flache aus Referenz-Elektrodenmaterial ist,
jedoch nicht an diese anstoGt, wobei die beiden
Elektrodenflachen klein dimensioniert sind und sich
als langliches Teil oder als vom langlich geformten
Elektrodentrager getragenes Teil erstrecken, urn die
Handhabung vor oder wahrend eines Kontaktes mit
Lebendgewebe oder mit einer kleinen Entnahme-
probe aus Korperflussigkert zu erleichtern, und
wobei das Arbeits-ElektrodenmateriaJ ein einschich-
tiges Gemisch des Glukoseenzyms, der Ferro-
cen-Vermittlerverbindung und leitfahigem Kohlen-
stoff ist.

2. Sensor-Elektrodensystem nach Anspruch 1, das
eine getrennte elektrische Verbindung zu jeder Elek-
trode fur einen AnschluB an ein das Vorhandensein,
die Menge oder den uberwachten Pegel der Glu-
kose in einem flussigen Medium anzeigenden Aus-
lesemittel, mit dem sich der Trager in Kontakt befin-
det, urn beide Elektroden in Kontakt zu bringen,
umfaBt.

9

17

EP0 127 958 B2

18

3. Sensor-Elektrodensystem nach Anspruch 2, mit

(a) einem flachen ersten Elektrodenbereich
bekannter Flache, die klein genug ist, urn voll-
standig durch den Blutabstrich bedeckt zu wer-
den, der von einem nicht herausgedruckten
Blutstropfen stammt, welcher durch einen
Nadelstich in eine Korperextremitat entnom-
men wird,

(b) einer auf derselben Oberflache angeordne-
ten Referenz-Elektrodenflache, die zwar
getrennt, aber doch so nan zur empfindlichen
Elektrodenflache liegt, daB der Blutabstrich
auch die Referenz-Elektrode erreicht, um eine
elektrische Verbindung herzustellen und

(c) getrennten, leitfahigen Elementen, die sich
entlang der gleichen Oberflache des langlichen
Tragerteiles erstrecken, wobei jeweils eines mit
jeder Elektrode fur einen AnschluB an
Signal-Auslesemittel, die an einem Ende des
Teiles angebracht werden konnen, verbunden
ist.

4. Sensor-Elektrodensystem nach Anspruch 1 , 2 oder
3, dadurch gekennzeichnet, daB die Flache aus
Arbeits-Elektrodenmaterial quadratisch ist.

5. Sensor-Elektrodensystem nach einem der Ansp ru-
che 1 bis 4, dadurch gekennzeichnet, daft die Fla-
che aus Arbeits-Elektrodenmaterial kieiner als 25
mm 2 ist.

6. Sensor-Elektrodensystem nach einem der vorste-
henden Anspruche, dadurch gekennzeichnet, daB
die Referenz-Elektrode aus Silber/Silberchlorid ist.

7. Sensor-Elektrodensystem nach Anspruch 1,
dadurch gekennzeichnet, daB die Arbeits-Elektrode
und die Referenz-Elektrode auf derselben Flache
eines langlichen nichtleitlahigen Tragers rechtecki-
gen Querschnittes aufgebracht sind.

8. Sensor-Elektrodensystem nach Anspruch 7,
dadurch gekennzeichnet, daB die Flache des
Arbeits-Elektrodenmaterials zu einem Ende der
Oberflache des langlichen Teiles hin angeordnet ist,
und daB die Flache der Referenz-Elektrode eben-
falls zu diesem Ende hin angeordnet ist.

Revendications

transfere des Electrons entre une electrode et
T enzyme oxydoreductase catalytiquement active,
caracteris6 en ce que le system e d'electrodes pour
detecteurs comprend un porteur effect rode sous

5 forme d'une languette test a jeter comportant une
zone de matiere formant electrode de travail com-
prenant 1'enzyme de glucose et le ferrocene ou un
derive de ferrocene comme le compose m6diateur,
la zone etant adjacente mais non contigue a une

io zone de matiere formant Electrode de reference, les
deux zones d'electrodes etant de faible dimensions
et s'etendant sur ou support6es par le porteur d'elec-
trodes qui se presente sous la forme d'un element
allonge de maniere a faciliter la manipulation avant

is ou pendant le contact avec le tissu vivant ou avec
un petit echantillon preleve de fluide corporel, dans
lequel ladit matiere formant electrode de travail est
un melange de ladite enzyme de glucose, ledit fer-
rocene et du carbone conducteur.

20

2. Un systeme d'electrodes pour detecteurs suivant la
revendication 1, comprenant une connexion electri-
que separee a chaque electrode pour un raccorde-
ment a un dispositif de sortie des donnees de
25 mesure montrant, la quantity ou le taux contr6le de
la glucose dans un milieu liquide avec lequel Pele-
ment support est mis en contact de maniere a assu-
rer le contact avec chacune des electrodes.

30 3. Un systeme d'electrodes pour detecteurs suivant la
revendication 2, qui comprend

(a) une premiere zone d'electrode plane de
zone connue suffisamment petite pour pouvoir

35 etre complement couverte par la tache de

sang produite par une goutte non exprimee de
sang provenant d'une piqure d'aiguille a une
extremite corporelle et

(b) une zone d'electrode de reference sur la
40 meme surface mais s6par6e de tout en etant

suffisamment proche de la zone d'electrode
sensible de sorte que ladite tache de sang
atteint egalement I'electrode de reference pour
etablir une communication electrique et

45 (c) des elements conducteurs separes s'eten-

dant le long de la meme surface de Tenement
support allonge, communiquant chacun avec
une electrode pour assurer la connexion avec
un dispositif de reception des donnees de

50 mesure pouvant etre attache a une extremite du

membre.

Un systeme d'electrodes pour detecteurs du genre
destine a une utilisation dans la detection, la mesure
ou le contrdle de glucose dans une melange liquide ss
en controlant le debit de courant lorsqu'une enzyme
oxydoreductase de glucose catalyse une reaction
redox de glucose et qu'un compose mediate ur

4. Un systeme d'electrodes pour detecteurs suivant la
revendication 1, 2 ou 3, dans lequel la zone de la
matiere formant electrode de travail est carr6e.

5. Un systeme d'electrodes pour detecteurs suivant
I'une quelconque des revendications 1 a 4, dans

10

19

EP0127 958 B2

lequel la zone de la matiere formant electrode de
travail est inf eneur a 25 mm 2 .

Un systeme d'electrodes pour de-tecteurs suivant
Tune quelconque des revendications precedent es, s
dans lequel I'Slectrode de reference est une elec-
trode argent/chlorure d'argent.

Un systeme d'electrodes pour detecteurs suivant la
revendicatlon 1 , dans lequel I'electrode de travail et io
Pelectrode de reference sont supportees sur la
meme face d'un support al)ong6 non conducteur de
section transversale rectangulaire.

, Un systeme d'electrodes pour d6tecteurs suivant la is
revendication 7, dans lequel la zone de la matiere
formant electrode de travail est situSe vers une
extremite de la surface de Tenement allonge et la
zone de I'electrode de reference est 6galement
situ6e vers la meme extremity 20

11

EP0127 958 B2

FIG.1

/

3
8

FIG. 2

21 FIG.3.

12

EP0127 958 B2

13

EP0127 958 B2

Z 8 ' ! 9 «

REFERENCE

VOLTAGE
BUFFER

\9t

LCW

>

PASS

FILTER

102

FIG.9

OIVIOER

109

-A

111

TIMER

Ls158
\ 57

155

| [156

—

O.V.M.

104

DISPLAY

OIVIOER

101

Fie.ii

TIMER

14

EP0127 958 B2

15

EP0127 958 B2

rm n

16